Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars
© 2014, Springer-Verlag Berlin Heidelberg. The β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous is the largest microbial enzyme producing neo-fructooligosaccharides (neo-FOS) known to date. It mainly synthesizes neokestose and neonystose, oligosaccharides with potentially imp...
| Autores: | , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2015 |
| País: | España |
| Institución: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/139327 |
| Acceso en línea: | http://hdl.handle.net/10261/139327 |
| Access Level: | acceso abierto |
| Palabra clave: | Extracellularβ-fructofuranosidase Blastose Pichia pastoris Heterologous expression Neo-fructooligosaccharides Xanthophyllomyces dendrorhous |
| Sumario: | © 2014, Springer-Verlag Berlin Heidelberg. The β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous is the largest microbial enzyme producing neo-fructooligosaccharides (neo-FOS) known to date. It mainly synthesizes neokestose and neonystose, oligosaccharides with potentially improved prebiotic properties. The Xd-INV gene comprises an open reading frame of 1995 bp, which encodes a 665-amino acid protein. Initial N-terminal sequencing of Xd-INV pointed to a majority extracellular protein of 595 amino acids lacking the first 70 residues (potential signal peptide). Functionality of the last 1785 bp of Xd-INV gene was previously proved in Saccharomyces cerevisiae but only weak β-fructofuranosidase activity was quantified. In this study, different strategies to improve this enzyme level in a heterologous system have been used. Curiously, best results were obtained by increasing the protein N-terminus sequence in 39 amino acids, protein of 634 residues. The higher β-fructofuranosidase activity detected in this study, about 15 U/mL, was obtained using Pichia pastoris and represents an improvement of about 1500 times the level previously obtained in a heterologous organism and doubles the best level of activity obtained by the natural producer. Heterologously expressed protein was purified and characterized biochemically and kinetically. Except by its glycosylation degree (10 % lower) and thermal stability (4–5 °C lower in the 60–85 °C range), the properties of the heterologous enzyme, including ability to produce neo-FOS, remained unchanged. Interestingly, besides the neo-FOS referred before blastose was also detected (8–22 g/L) in the reaction mixtures, making Xd-INV the first yeast enzyme producing this non-conventional disaccharide reported to date. |
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